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PURPOSE OF REVIEW: Environmental fungi such as Cryptococcus neoformans and Aspergillus fumigatus must survive many different and changing environments as they transition from their environmental niches to human lungs and other organs. Fungi alter their cell surfaces and secreted macromolecules to respond to and manipulate their surroundings. RECENT FINDINGS: This review focuses on exo-polysaccharides, chains of sugars that transported out of the cell and spread to the local environment. Major exo-polysaccharides for C. neoformans and A. fumigatus are glucuronylxylomannan (GXM) and galactosaminogalactan (GAG), respectively, which accumulate at high concentrations in growth medium and infected patients SUMMARY: Here we discuss GXM and GAG synthesis and export, their immunomodulatory properties, and their roles in biofilm formation. We also propose areas of future research to address outstanding questions in the field that could facilitate development of new disease treatments.

Environmental fungi are globally ubiquitous and human exposure is near universal. However, relatively few fungal species are capable of infecting humans, and among fungi, few exposure events lead to severe systemic infections. Systemic infections have mortality rates of up to 90%, cost the US healthcare system $7.2 billion annually, and are typically associated with immunocompromised patients. Despite this reputation, exposure to environmental fungi results in a range of outcomes, from asymptomatic latent infections to severe systemic infection. Here we discuss different exposure outcomes for five major fungal pathogens: Aspergillus, Blastomyces, Coccidioides, Cryptococcus, and Histoplasma species. These fungi include a mold, a budding yeast, and thermal dimorphic fungi. All of these species must adapt to dramatically changing environments over the course of disease. These dynamic environments include the human lung, which is the first exposure site for these organisms. Fungi must defend themselves against host immune cells while germinating and growing, which risks further exposing microbe-associated molecular patterns to the host. We discuss immune evasion strategies during early infection, from disruption of host immune cells to major changes in fungal cell morphology.

SUMMARYEnvironmental pathogens, which move from ecological niches to mammalian hosts, must adapt to dramatically different environments. Microbes that disseminate farther, including the fungal meningitis pathogen Cryptococcus neoformans, require additional adaptation to diverse tissues. When C. neoformans enters the lungs, infecting cells (30 μm diameter), then form a heterogeneous population. The brain contains uniformly small cells (∼7 μm). We demonstrate that formation of a small C. neoformans morphotype – called “seed” cells due to their disseminating ability – is critical for extrapulmonary organ entry. Seed cell formation is triggered by environmental factors, including C. neoformans’ environmental niche, pigeon guano. The underlying trigger, phosphate, can be released by tissue damage, potentially establishing a feed-forward loop of seed cell formation and dissemination. We demonstrate that C. neoformans’ size variation is not just a continuum but inducible subpopulations that change host interactions to facilitate microbe survival and spread.

Invasive fungal infections cause 1.6 million deaths annually, primarily in immunocompromised individuals. Mortality rates are as high as 90% due to limited treatments. The azole class antifungal, fluconazole, is widely available and has multi-species activity but only inhibits growth instead of killing fungal cells, necessitating long treatments. To improve treatment, we used our novel high-throughput method, the overlap2 method (O2M) to identify drugs that interact with fluconazole, either increasing or decreasing efficacy. We identified 40 molecules that act synergistically (amplify activity) and 19 molecules that act antagonistically (decrease efficacy) when combined with fluconazole. We found that critical frontline beta-lactam antibiotics antagonize fluconazole activity. A promising fluconazole-synergizing anticholinergic drug, dicyclomine, increases fungal cell permeability and inhibits nutrient intake when combined with fluconazole. In vivo, this combination doubled the time-to-endpoint of mice with Cryptococcus neoformans meningitis. Thus, our ability to rapidly identify synergistic and antagonistic drug interactions can potentially alter the patient outcomes., eLife digest Individuals with weakened immune systems – such as recipients of organ transplants – can fall prey to illnesses caused by fungi that are harmless to most people. These infections are difficult to manage because few treatments exist to fight fungi, and many have severe side effects. Antifungal drugs usually slow the growth of fungi cells rather than kill them, which means that patients must remain under treatment for a long time, or even for life. One way to boost efficiency and combat resistant infections is to combine antifungal treatments with drugs that work in complementary ways: the drugs strengthen each other’s actions, and together they can potentially kill the fungus rather than slow its progression. However, not all drug combinations are helpful. In fact, certain drugs may interact in ways that make treatment less effective. This is particularly concerning because people with weakened immune systems often take many types of medications. Here, Wambaugh et al. harnessed a new high-throughput system to screen how 2,000 drugs (many of which already approved to treat other conditions) affected the efficiency of a common antifungal called fluconazole. This highlighted 19 drugs that made fluconazole less effective, some being antibiotics routinely used to treat patients with weakened immune systems. On the other hand, 40 drugs boosted the efficiency of fluconazole, including dicyclomine, a compound currently used to treat inflammatory bowel syndrome. In fact, pairing dicyclomine and fluconazole more than doubled the survival rate of mice with severe fungal infections. The combined treatment could target many species of harmful fungi, even those that had become resistant to fluconazole alone. The results by Wambaugh et al. point towards better treatments for individuals with serious fungal infections. Drugs already in circulation for other conditions could be used to boost the efficiency of fluconazole, while antibiotics that do not decrease the efficiency of this medication should be selected to treat at-risk patients.

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

Cryptococcus neoformansis a common environmental yeast and opportunistic pathogen responsible for 15% of AIDS-related deaths worldwide. Mortality primarily results from meningoencephalitis, which occurs when fungal cells disseminate from the initial pulmonary infection site and spread to the brain. A keyC. neoformansvirulence trait is the polysaccharide capsule. Capsule shields C. neoformans from immune-mediated recognition and destruction. The main capsule component, glucuronoxylomannan (GXM), is found both attached to the cell surface and free in the extracellular space (as exo-GXM). Exo-GXM accumulates in patient serum and cerebrospinal fluid at μg/mL concentrations, has well-documented immunosuppressive properties, and correlates with poor patient outcomes. However, it is poorly understood whether exo-GXM release is regulated or the result of shedding during normal capsule turnover. We demonstrate that exo-GXM release is regulated by environmental cues and inversely correlates with surface capsule levels. We identified genes specifically involved in exo-GXM release that do not alter surface capsule thickness. The first mutant,liv7∆, released less GXM than wild-type cells when capsule is not induced. The second mutant,cnag_00658∆, released more exo-GXM under capsule-inducing conditions. Exo-GXM release observedin vitrocorrelated with polystyrene adherence, virulence, and fungal burden during murine infection. Additionally, we find that exo-GXM reduces cell size and capsule thickness in capsule-inducing conditions, potentially influencing dissemination. Finally, we demonstrated that exo-GXM prevents immune cell infiltration into the brain during disseminated infection and highly inflammatory intracranial infection. Our data suggest that exo-GXM performs a different role from capsule GXM during infection, altering cell size and suppressing inflammation.ImportanceCryptococcus neoformansis a leading cause of life-threatening meningoencephalitis in humans.C. neoformanscells produce an immunosuppressive polysaccharide, glucuronoxylomannan (GXM), that is the main component of a protective surface capsule. GXM is also released free into extracellular space as exo-GXM, although the distinction between cell-attached GXM and exo-GXM has been unclear. Exo-GXM influences the outcome of infection, is the basis for current diagnostic tools, and has potential therapeutic applications. This study increases our basic understanding of the fungal biology that regulates polysaccharide release, suggesting that the release of cell-attached GXM and exo-GXM are distinctly regulated. We also introduce a new concept that exo-GXM may alter cell body and capsule size, thereby influencing dissemination in the host. Finally, we provide experimental evidence to confirm clinical observations that exo-GXM influences inflammation during brain infection.

Cryptococcus neoformans is a common environmental saprophyte and human fungal pathogen that primarily causes disease in immunocompromised individuals. Similar to many environmentally acquired human fungal pathogens, C. neoformans initiates infection in the lungs. However, the main driver of mortality is invasive cryptococcosis leading to fungal meningitis. After C. neoformans gains a foothold in the lungs, a critical early step in invasion is transversal of the respiratory epithelium. In this review, we summarize current knowledge relating to pulmonary escape. We focus on fungal factors that allow C. neoformans to disseminate from the lungs via intracellular and extracellular routes.

The commensal microbiota has an important impact on host health, which is only beginning to be elucidated. Despite the presence of fungal, archaeal, and viral members, most studies have focused solely on the bacterial microbiota. Antibodies against the yeast Saccharomyces cerevisiae are found in some patients with Crohn’s disease (CD), suggesting that the mycobiota may contribute to disease severity. We report that S. cerevisiae exacerbated intestinal disease in a mouse model of colitis and increased gut barrier permeability. Transcriptome analysis of colon tissue from germ-free mice inoculated with S. cerevisiae or another fungus, Rhodotorula aurantiaca, revealed that S. cerevisiae colonization affected the intestinal barrier and host metabolism. A fecal metabolomics screen of germ-free animals demonstrated that S. cerevisiae colonization enhanced host purine metabolism, leading to an increase in uric acid production. Treatment with uric acid alone worsened disease and increased gut permeability. Allopurinol, a clinical drug used to reduce uric acid, ameliorated colitis induced by S. cerevisiae in mice. In addition, we found a positive correlation between elevated uric acid and anti-yeast antibodies in human sera. Thus, yeast in the gut may be able to potentiate metabolite production that negatively affects the course of inflammatory bowel disease.

Summary The fungal meningitis pathogen Cryptococcus neoformans is a central driver of mortality in HIV/AIDS. We report a genome-scale chemical genetic data map for this pathogen that quantifies the impact of 439 small-molecule challenges on 1,448 gene knockouts. We identified chemical phenotypes for 83% of mutants screened and at least one genetic response for each compound. C. neoformans chemical-genetic responses are largely distinct from orthologous published profiles of Saccharomyces cerevisiae , demonstrating the importance of pathogen-centered studies. We used the chemical-genetic matrix to predict novel pathogenicity genes, infer compound mode of action, and to develop an algorithm, O2M, that predicts antifungal synergies. These predictions were experimentally validated, thereby identifying virulence genes, a molecule that triggers G2/M arrest and inhibits the Cdc25 phosphatase, and many compounds that synergize with the antifungal drug fluconazole. Our work establishes a chemical-genetic foundation for approaching an infection responsible for greater than one-third of AIDS-related deaths. PaperClip

Little is known about the molecular processes that govern female gametophyte (FG) development and function, and few FG-expressed genes have been identified. We report the identification and phenotypic analysis of 31 new FG mutants in Arabidopsis. These mutants have defects throughout development, indicating that FG-expressed genes govern essentially every step of FG development. To identify genes involved in cell death during FG development, we analyzed this mutant collection for lines with cell death defects. From this analysis, we identified one mutant, gfa2, with a defect in synergid cell death. Additionally, the gfa2 mutant has a defect in fusion of the polar nuclei. We isolated the GFA2 gene and show that it encodes a J-domain-containing protein. Of the J-domain-containing proteins in Saccharomyces cerevisiae (budding yeast), GFA2 is most similar to Mdj1p, which functions as a chaperone in the mitochondrial matrix. GFA2 is targeted to mitochondria in Arabidopsis and partially complements a yeast mdj1 mutant, suggesting that GFA2 is the Arabidopsis ortholog of yeast Mdj1p. These data suggest a role for mitochondria in cell death in plants.

Antibiotic-resistant infections kill approximately 23,000 people and cost $20,000,000,000 each year in the United States alone despite the widespread use of small-molecule antimicrobial combination therapy. Antibiotic combinations typically have an additive effect: the efficacy of the combination matches the sum of the efficacies of each antibiotic when used alone. Small molecules can also act synergistically when the efficacy of the combination is greater than the additive efficacy. However, synergistic combinations are rare and have been historically difficult to identify. High-throughput identification of synergistic pairs is limited by the scale of potential combinations: a modest collection of 1,000 small molecules involves 1 million pairwise combinations. Here, we describe a high-throughput method for rapid identification of synergistic small-molecule pairs, the overlap2 method (O2M). O2M extracts patterns from chemical-genetic datasets, which are created when a collection of mutants is grown in the presence of hundreds of different small molecules, producing a precise set of phenotypes induced by each small molecule across the mutant set. The identification of mutants that show the same phenotype when treated with known synergistic molecules allows us to pinpoint additional molecule combinations that also act synergistically. As a proof of concept, we focus on combinations with the antibiotics trimethoprim and sulfamethizole, which had been standard treatment against urinary tract infections until widespread resistance decreased efficacy. Using O2M, we screened a library of 2,000 small molecules and identified several that synergize with the antibiotic trimethoprim and/or sulfamethizole. The most potent of these synergistic interactions is with the antiviral drug azidothymidine (AZT). We then demonstrate that understanding the molecular mechanism underlying small-molecule synergistic interactions allows the rational design of additional combinations that bypass drug resistance. Trimethoprim and sulfamethizole are both folate biosynthesis inhibitors. We find that this activity disrupts nucleotide homeostasis, which blocks DNA replication in the presence of AZT. Building on these data, we show that other small molecules that disrupt nucleotide homeostasis through other mechanisms (hydroxyurea and floxuridine) also act synergistically with AZT. These novel combinations inhibit the growth and virulence of trimethoprim-resistant clinical Escherichia coli and Klebsiella pneumoniae isolates, suggesting that they may be able to be rapidly advanced into clinical use. In sum, we present a generalizable method to screen for novel synergistic combinations, to identify particular mechanisms resulting in synergy, and to use the mechanistic knowledge to rationally design new combinations that bypass drug resistance., Author summary Antibiotic resistance is a growing problem that threatens our ability to treat systemic bacterial infections. One strategy to combat antibiotic resistance is the use of synergistic antibiotic pairs that, when combined, have activity that is considerably greater than the sum of each individual drug’s activity on its own. Synergistic combinations can even inhibit the growth of bacteria that are resistant to the individual treatment drugs. However, synergistic pairs are rare and difficult to identify. High-throughput identification of synergistic pairs is challenging due to scale: 1 million different pairs are possible for a relatively small collection of 1,000 small molecules. Here, we describe a high-throughput method for rapid identification of synergistic small-molecule pairs, termed the overlap2 method (O2M), that dramatically speeds up the screening process. First, we identify mutants that show the same phenotype when treated with each individual molecule in a synergistic pair, then use this information to guide screens for additional synergistic pairs. As a proof of concept, we studied the synergistic antibiotic pair trimethoprim and sulfamethizole, and we identified several additional synergistic molecules. Among these is the antiviral drug azidothymidine (AZT), which blocks bacterial DNA replication. Trimethoprim and sulfamethizole both inhibit folate biosynthesis, which is necessary for the proper synthesis of nucleotides for DNA replication and repair. We found that reduced nucleotide levels sensitize E. coli cells to AZT. When we substitute trimethoprim with other small molecules that also reduce nucleotide levels, we find that these small molecules also act synergistically with AZT. Indeed, AZT in combination with trimethoprim substitutes inhibits the growth of trimethoprim-resistant clinical isolates more potently than trimethoprim and AZT or trimethoprim and sulfamethizole. This work demonstrates that when we resolve the pathways that underlie synergistic interactions, we can then identify additional small molecules that act by similar mechanisms, providing a means to bypass antibiotic resistance.

In experimental science, organisms are usually studied in isolation, but in the wild they compete and cooperate in complex communities. We report a system for cross-kingdom communication by which bacteria heritably transform yeast metabolism. An ancient biological circuit blocks yeast from using other carbon sources in the presence of glucose. [GAR[superscript +]], a protein-based epigenetic element, allows yeast to circumvent this glucose repression and use multiple carbon sources in the presence of glucose. Some bacteria secrete a chemical factor that induces [GAR[superscript +]]. [GAR[superscript +]] is advantageous to bacteria because yeast cells make less ethanol, and is advantageous to yeast because their growth and long-term viability is improved in complex carbon sources. This crosskingdom communication is broadly conserved, providing a compelling argument for its adaptive value. By heritably transforming growth and survival strategies in response to the selective pressures of life in a biological community, [GAR[superscript +]] presents a unique example of Lamarckian inheritance., G. Harold and Leila Y. Mathers Foundation, Howard Hughes Medical Institute

The aflatoxin biosynthetic pathway represents one of the est studied pathways of fungal secondary metabolism. Its lucidation is the result of over 30 years of study by cientists in many disciplines. For recent reviews on the hemistry of the pathway see articles by Bhatnagar et al. 1992) Minto and Townsend (1997), and Woloshuk and rieto (1997). Concern over the toxicity and carcinogenicty of aflatoxin has been the prime force driving research in his area. Aflatoxin B1 (AFB1) is the most potent naturally ccurring carcinogen known (Squire, 1989), and epidemioogical data implicate aflatoxin as a component of liver ancer in humans in certain parts of the world (Hall and ild, 1994). Although aflatoxins are not extremely toxic, onsumption of aflatoxin contaminated food by animals can ead to decreased weight gain, hemorrhaging, and suppression f the immune system (Miller and Wilson, 1994). Aflatoxins are produced by four species of Aspergillus: . flavus, A. parasiticus, A. nomius, and A. tamarii (Payne, 998; Kurtzman et al., 1986; Goto et al., 1996). From an gricultural standpoint, the two most significant fungi are

[GAR[superscript +]] is a protein-based element of inheritance that allows yeast (Saccharomyces cerevisiae) to circumvent a normal hallmark of their biology: extreme metabolic specialization for glucose fermentation. When glucose is present, even in trace quantities, yeast will not use other carbon sources. [GAR[superscript +]] allows cells to circumvent this “glucose repression.” [GAR[superscript +]] is induced in yeast by a factor secreted by bacteria inhabiting their environment. We report that the de novo rates of [GAR[superscript +]] appearance correlate with the yeast’s ecological niche. Evolutionarily distant fungi possess similar epigenetic elements that are also induced by bacteria. As expected for a mechanism whose adaptive value originates from the selective pressures of life in biological communities, the ability of bacteria to induce [GAR[superscript +]] and the ability of yeast to respond to bacterial signals have been extinguished repeatedly during the extended monoculture of domestication. Thus, [GAR[superscript +]] is a broadly conserved adaptive strategy that links environmental and social cues to heritable changes in metabolism., G. Harold and Leila Y. Mathers Foundation, Howard Hughes Medical Institute

Four bacteriophages (phages) growing on the same Leuconostoc strain were characterised. Electron micrographs showed these phages to be similar in morphology to the commonly isolated lactococcal phages with head diameters ranging from 49–55 nm and tail lengths of 117–131 nm. A distinctive base plate and collar were also present. From restriction enzyme analysis of purified phage DNA, the genome sizes were 23–29 kb. All four phages showed one major structural protein (of approximately 24 kDa) on SDS polyacrylamide gels. Hybridization experiments confirmed that the phages belonged to the same homology group. There was no homology between DNA from these phages and DNA from a prolate or small isometric lactococcal phage.

This paper reports on the application of the ligase chain reaction (LCR) to the specific detection of variants of the nisin structural gene (nisinA and nisinZ) in nisin producing strains of Lactococcus lactis ssp lactis. The LCR assay was used to screen nisin producing strains to determine which form of the nisin structural gene they contained. This method of differentiating the nisin structural gene variants provides a useful alternative to the only other available genetic differentiation, that of sequencing the gene.

Chimeric proteins consisting of the VP2 capsid protein of human parvovirus B19 and defined linear epitopes from human herpes simplex virus type 1 and mouse hepatitis virus A59 inserted at the N-terminus and at a predicted surface region were expressed by recombinant baculoviruses. The chimeric proteins expressed the inserted epitopes and assembled into empty capsids. Immunoelectron microscopy indicated that the epitopes inserted in the loop were exposed on the surface of the chimeric particles. The chimeric capsids were immunogenic in mice and antibodies specific for the inserted sequences were induced. In the case of MHV, antibodies were produced that recognized the epitope in the context of native virus. Mice immunized with the chimeric capsids were partially protected against a lethal challenge infection with either MHV or HSV.

An international collaborative study was conducted at ten sites to examine the performance of enzyme immunoassays (EIAs) for the quantitation of IgG1, IgG2, IgG3, IgG4 and total IgG anti-Haemophilus influenzae type b (Hib) capsular polysaccharide in human serum. All groups used the same reagents: microtiter plates coated with polyribosylribitol phosphate (PRP) conjugated to poly-L-lysine (PLL), reference, control and test human sera, biotin-conjugated International Union of Immunological Societies (IUIS)-documented monoclonal anti-human IgG1-4 and IgG Pan detection antibodies, avidin-peroxidase and TMB substrate. Initial mixing of soluble PRP antigen or an equal volume of buffer with the 20 test sera prior to analysis confirmed PRP antigen specificity in all five EIAs with greater than 80% competitive inhibition at most sites. Positive correlation between the total IgG anti-Hib and sum of IgG1-4 anti-Hib was demonstrated (r2 = 0.99, Y = 1.13X -0.15). Good agreement was shown between the total IgG anti-Hib as measured by EIA and the total Hib-specific antibodies measured by the current radiolabeled antigen binding assay (r2 = 0.97, Y = 4.6X -5.8). Assay parallelism was demonstrated with an average interdilutional %CV of 22% and parallel dose-response curve slopes. The interdilutional %CVs were calculated as an average per sample of the variation of microgram/ml (corrected for dilution) at different dilutions per laboratory for all participating sites. The interlaboratory variation was the only performance parameter studied that exceeded the target level of 35% CV in all IgG1-4 and total IgG anti-Hib assays. IgG subclass distributions in the test sera demonstrated a predominance of IgG1 anti-Hib in the pediatric serum pools and IgG2 anti-Hib in the adult sera, with low but detectable levels of IgG3 and IgG4 anti-Hib in each group.

Several well-characterized fungal proteins act as prions, proteins capable of multiple conformations, each with different activities, at least one of which is self-propagating. Through such self-propagating changes in function, yeast prions act as protein-based elements of phenotypic inheritance. We report a prion that makes cells resistant to the glucose-associated repression of alternative carbon sources, [GAR[superscript +]] (for “resistant to glucose-associated repression,” with capital letters indicating dominance and brackets indicating its non-Mendelian character). [GAR[superscript +]] appears spontaneously at a high rate and is transmissible by non-Mendelian, cytoplasmic inheritance. Several lines of evidence suggest that the prion state involves a complex between a small fraction of the cellular complement of Pma1, the major plasma membrane proton pump, and Std1, a much lower-abundance protein that participates in glucose signaling. The Pma1 proteins from closely related Saccharomyces species are also associated with the appearance of [GAR[superscript +]]. This allowed us to confirm the relationship between Pma1, Std1, and [GAR[superscript +]] by establishing that these proteins can create a transmission barrier for prion propagation and induction in Saccharomyces cerevisiae. The fact that yeast cells employ a prion-based mechanism for heritably switching between distinct carbon source utilization strategies, and employ the plasma membrane proton pump to do so, expands the biological framework in which self-propagating protein-based elements of inheritance operate., United States. National Institutes of Health (grant GM25874)

The large subunit of Saccharomyces cerevisiae DNA polymerase epsilon, Pol2, comprises two essential functions. The N terminus has essential DNA polymerase activity. The C terminus is also essential, but its function is unknown. We report here that the C-terminal domain of Pol2 interacts with polymerase sigma (Pol sigma), a recently identified, essential nuclear nucleotidyl transferase encoded by two redundant genes, TRF4 and TRF5. This interaction is functional, since Pol sigma stimulates the polymerase activity of the Pol epsilon holoenzyme significantly. Since Trf4 is required for sister chromatid cohesion as well as for completion of S phase and repair, the interaction suggested that Pol epsilon, like Pol sigma, might form a link between the replication apparatus and sister chromatid cohesion and/or repair machinery. We present evidence that pol2 mutants are defective in sister chromatid cohesion. In addition, Pol2 interacts with SMC1, a subunit of the cohesin complex, and with ECO1/CTF7, required for establishing sister chromatid cohesion; and pol2 mutations act synergistically with smc1 and scc1. We also show that trf5 Delta mutants, like trf4 Delta mutants, are defective in DNA repair and sister chromatid cohesion.

Cisplatin (CDDP) has been used as a DNA cross-linking agent to evaluate whether there is a specific cell cycle checkpoint response to such damage in Saccharomyces cerevisiae (S. cerevisiae). Fluorescent-activated cell sorting (FACS) analysis showed only a G2/M checkpoint, normal exit from G1 and progression through S-phase following alpha-factor arrest and CDDP treatment. Of the checkpoint mutants tested, rad9, rad17 and rad24, did not show increased sensitivity to CDDP compared to isogenic wild-type cells. However, other checkpoint mutants tested (mec1, mec3 and rad53) showed increased sensitivity to CDDP, as did controls with a defect in excision repair (rad1 and rad14) or a defect in recombination (rad51 and rad52). Thus, by survival and cell cycle kinetics, it appears that DNA cross-links do not inhibit entry into S-phase or slow DNA replication and that replication continues after cisplatin treatment in yeast.

The 16S ribosomal RNA gene sequences of Lactococcus lactis ssp. lactis and ssp. cremoris differ by 9–10 bp (depending on strain), within the first 200 bp of the sequence. These differences were used to develop two methods of genetically differentiating lactis and cremoris strains. Primers to conserved sequences in the 16S rRNA gene were used in a PCR reaction to amplify fragments of the 16S rRNA gene. A single base difference at position 180 of the sequence was utilised to develop a ligase chain reaction to differentiate lactis and cremoris sequences. The second method involved digestion of the amplified fragments with restriction endonucleases specific for either the lactis or cremoris sequence. Resolution of the digested fragments on an agarose gel allowed the strains to be identified as genetically lactis or cremoris. This method was used to examine lactococci isolated from raw milk. Of 31 raw milk strains examined, 21 contained the cremoris 16S rRNA sequence, however, all 31 strains exhibited the phenotypic characteristics of the lactis subspecies.

In many human fungal pathogens, genes required for disease remain largely unannotated, limiting the impact of virulence gene discovery efforts. We tested the utility of a cross-species genetic interaction profiling approach to obtain clues to the molecular function of unannotated pathogenicity factors in the human pathogen Cryptococcus neoformans. This approach involves expression of C. neoformans genes of interest in each member of the Saccharomyces cerevisiae gene deletion library, quantification of their impact on growth, and calculation of the cross-species genetic interaction profiles. To develop functional predictions, we computed and analyzed the correlations of these profiles with existing genetic interaction profiles of S. cerevisiae deletion mutants. For C. neoformans LIV7, which has no S. cerevisiae ortholog, this profiling approach predicted an unanticipated role in the Golgi apparatus. Validation studies in C. neoformans demonstrated that Liv7 is a functional Golgi factor where it promotes the suppression of the exposure of a specific immunostimulatory molecule, mannose, on the cell surface, thereby inhibiting phagocytosis. The genetic interaction profile of another pathogenicity gene that lacks an S. cerevisiae ortholog, LIV6, strongly predicted a role in endosome function. This prediction was also supported by studies of the corresponding C. neoformans null mutant. Our results demonstrate the utility of quantitative cross-species genetic interaction profiling for the functional annotation of fungal pathogenicity proteins of unknown function including, surprisingly, those that are not conserved in sequence across fungi., Author Summary HIV/AIDS patients, cancer chemotherapy patients, and organ transplant recipients are highly susceptible to infection by opportunistic fungal pathogens, organisms common in the environment that are harmless to normal individuals. Understanding how these pathogens cause disease requires the identification of genes required for virulence and the determination of their molecular function. Our work addresses the latter problem using the yeast Cryptococcus neoformans, which is estimated to cause 600,000 deaths annually worldwide in the HIV/AIDS population. We describe a method for determining gene function in which C. neoformans genes are expressed in deletion mutants of all nonessential genes of the well-studied model yeast S. cerevisiae. By examining the impact on growth (enhancement or suppression) we generated “cross-species” genetic interaction profiles. We compared these profiles to the published genetic interaction profiles of S. cerevisiae deletion mutants to identify those with correlated patterns of genetic interactions. We hypothesized that the known functions of S. cerevisiae genes with correlated profiles could predict the function of the pathogen gene. Indeed, experimental tests in C. neoformans for two pathogenicity genes of previously unknown function found the functional predictions obtained from genetic interaction profiles to be accurate, demonstrating the utility of the cross-species approach.

An immunodominant region on baculovirus-produced parvovirus B19 VP2 capsids was localized between amino acids 259 and 426 by mapping the binding sites of a panel of monoclonal antibodies which recognize determinants on the particles. The binding sites of three monoclonal antibodies were fine-mapped within this antigenic domain. Six VP2-specific monoclonal antibodies recognized determinants common to both the empty capsids and native parvovirus. The defined antigenic region is most probably exposed on the native B19 virion and corresponds to part of the threefold spike on the surface of canine parvovirus particles.

Empty parvovirus B19 capsids were isolated from insect cells infected with a recombinant baculovirus expressing parvovirus B19 VP2 alone and also with a double-recombinant baculovirus expressing both VP1 and VP2. That VP2 alone can assemble to form capsids is a phenomenon not previously observed in parvoviruses. The stoichiometry of the capsids containing both VP1 and VP2 was similar to that previously observed in parvovirus B19-infected cells. The capsids were similar to native capsids in size and appearance, and their antigenicity was demonstrated by immunoprecipitation and enzyme-linked immunosorbent assay with B19-specific antibodies.

B. Dresser’,‘, E. Pope’,*, N. Chin3, J. Liu*, N. Loskutoff4, E. Behnke3, C. Brown4, M. McRae’, C. Sinoway3, M. Campbell’, K. Cameron’, R. Evans’, 0. 0wens3, C. Johnson’, M. Cedars* ‘Cincinnati Zoo Center for Reproduction of Endangered Wildlife (CREW), 3400 Vine Street, Cincinnati, OH 45220 USA; *University of Cincinnati College of Medicine, Department of Obstetrics and Gynecology, Cincinnati, OH; 3The Christ Hospital, Cincinnati, OH; 40maha’s Henry Doorly Zoo, Omaha, NE; 5Bethesda Hospital, Cincinnati, OH.

Human red blood cells (RBCs), transformed by incubation with the amphiphatic compound lysolecithin from their normal discocyte shape into echinocytes, have increased rates of agglutination in the presence of either poly- l -lysine (PLL) or soybean agglutinin (SBA). Removal of lysolecithin by washing caused a reversal of shape back to the discocyte configuration and a lowering of agglutination rates. Methochlorpromazine, another amphiphatic echinocytogenic substance produced a similar increase in agglutination rates, suggesting that increased agglutinability may be a general property of echinocytes. Lysolecithin treatment of RBCs caused a decrease in the binding of cationized ferritin (CF) particles/μm2 of RBC surface. The decrease in CF binding is due to a rearrangement of negative charge bearing molecules on the RBC surface rather than shedding of charged groups. These observations support the hypothesis that integral membrane proteins which bear negative charges and receptors are associated with a cytoskeleton within the red cell. Alterations in cell shape which result in distortion of the cytoskeleton may cause a redistribution of integral membrane proteins which bear charged groups at the RBC surface.

SummaryThe antiphagocytic polysaccharide capsule of the human fungal pathogen Cryptococcus neoformans is a major virulence attribute. However, previous studies of the pleiotropic virulence determinant Gat201, a GATA family transcription factor, suggested that capsule-independent antiphagocytic mechanisms exist. We have determined that Gat201 controls the mRNA levels of ∼1100 genes (16% of the genome) and binds the upstream regions of ∼130 genes. Seven Gat201-bound genes encode for putative and known transcription factors—including two previously implicated in virulence—suggesting an extensive regulatory network. Systematic analysis pinpointed two critical Gat201-bound genes, GAT204 (a transcription factor) and BLP1, which account for much of the capsule-independent antiphagocytic function of Gat201. A strong correlation was observed between the quantitative effects of single and double mutants on phagocytosis in vitro and on host colonization in vivo. This genetic dissection provides evidence that capsule-independent antiphagocytic mechanisms are pivotal for successful mammalian infection by C. neoformans.